The observation of samples, and in particular biological samples, by lens-free imaging has seen substantial development over the last ten years. This technique allows a sample placed between a light source and an image sensor to be observed without a magnifying optical lens placed between the sample and the image sensor. Thus, the image sensor collects an image of the light wave transmitted by the sample.
This image is made up of interference patterns formed by interference between the light wave emitted by the light source and transmitted by the sample, and diffracted waves resulting from the diffraction by the sample of the light wave emitted by the light source. These interference patterns are sometimes denoted diffraction patterns.
Document WO2008090330 describes a device allowing biological samples, in this case cells, to be observed by lens-free imaging. The device allows an interference pattern to be associated with each cell and the morphology of this pattern allows the type of cell to be identified. Lens-free imaging would thus appear to be a simple and inexpensive alternative to conventional microscopy. In addition, its field of observation is clearly larger than that obtainable with a microscope. Thus, it will be understood that the potential applications of this technology are broad and far-reaching.
Generally, the image formed on the image sensor, i.e. the interference pattern image, may be processed by way of a holographic reconstruction algorithm, so as to estimate optical properties of the sample. Such algorithms are well known in the field of holographic reconstruction. To do this, the distance between the sample and the image sensor being known, a propagation algorithm, taking into account this distance and the wavelength of the light wave emitted by the light source, is applied. It is then possible to create an image of an optical property of the sample. A numerical reconstruction algorithm is for example described in US 2012/0218379.
The relevance of lens-free imaging to histological analysis of cancerous tissue samples taking the form of a thin strip, or slice, deposited on a transparent holder has recently been demonstrated. The large field of observation achieved with lens-free imaging, coupled to its low cost, are decisive advantages with respect to the use of a conventional microscope. In addition, the use of holographic reconstruction algorithms makes it possible to form an absorption image or a phase image of the observed sample of a high enough quality for a reliable histological analysis to be carried out.
The publication Greenbaum A, “wide-field computational imaging of pathology slides using lens-free on-chip microscopy”, Sci. Transl. Med 6, 267ra175 (2014), for example describes the application of lens-free imaging to anatomopathological slides formed by sampling various types of biological tissues. The same is also true of the publication Luo W. “Synthetic aperture-based on-chip microscopy”, Light: Science & Applications (2015) 4, e261. In this publication, an optical coupling oil is applied between the image sensor and the sample so as to improve the optical coupling. However, the time required to obtain an image of a sample of 1 mm2 exceeds 45 minutes, this being far too long and incompatible with routine use.
The inventors provide a method for observing a sample, and in particular a tissue sample, by lens-free imaging, implementing holographic reconstruction methods and allowing a clearer image to be formed. This method is particularly suitable for the observation of biological tissues.